On the Utility of Chemical Strategies to Improve Peptide Gut Stability

Inherent susceptibility of peptides to enzymatic degradation in the gastrointestinal tract is a key bottleneck in oral peptide drug development. Here, we present a systematic analysis of the gut stability of disulfide-rich peptide scaffolds, orally administered peptide therapeutics, and well-known neuropeptides and medicinal chemistry strategies to improve peptide gut stability. Among a broad range of studied peptides, cyclotides were the only scaffold class to resist gastrointestinal degradation, even when grafted with non-native sequences. Backbone cyclization, a frequently applied strategy, failed to improve stability in intestinal fluid, but several site-specific alterations proved efficient. This work furthermore highlights the importance of standardized gut stability test conditions and suggests defined protocols to facilitate cross-study comparison. Together, our results provide a comparative overview and framework for the chemical engineering of gut-stable peptides, which should be valuable for the development of orally administered peptide therapeutics and molecular probes targeting receptors within the gastrointestinal tract.

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About this article: On the Utility of Chemical Strategies to Improve Peptide Gut Stability – Thomas Kremsmayr, Aws Aljnabi, Juan B. Blanco-Canosa, Hue N. T. Tran, Nayara Braga Emidio, and Markus Muttenthaler – Journal of Medicinal Chemistry Article ASAP – DOI: 10.1021/acs.jmedchem.2c00094

Conclusion
In conclusion, this comprehensive gut stability analysis of a series of representative disulfide-rich peptide classes including natural scaffolds, therapeutic leads, neuropeptides, and approved peptide drugs provided several new insights and guidance for the development of gut-stable peptides. We demonstrated that only few native scaffolds and chemical modifications resisted degradation in the intestinal environment, including those that previously demonstrated high stability in other media (i.e., serum, single digestive enzymes). For instance, backbone cyclization, a frequently proposed medicinal chemistry approach to improve peptide stability, provided no tangible metabolic protection against intestinal degradation. By contrast site-specific peptide backbone modifications via Cα- or Nα-methylation, β3-homo amino acid and d-amino acids effectively prevented intestinal metabolism. Most natural cyclic and disulfide-rich scaffolds were not stable in intestinal fluid, particularly once modified with non-native sequences. The exception was the ICK class of plant-based cyclotides, likely because of their defense role in deterring animals eating their plant hosts, which retained evolutionarily optimized high gut stability even with non-native sequences incorporated. This renders cyclotides highly promising as gut-stable vectors to deliver therapeutic sequences inside the gut lumen. Taken together, our results provide a comparative framework and novel insights to support the development of gut-stable peptides, a highly important undertaking given the vast therapeutic potential of orally administered peptides for gut-specific action.